Alpha-synuclein is a major protein component of Lewy bodies, a cardinal feature of the degenerating Parkinsonian brain. Alpha-synuclein has been demonstrated to intercalate into lipid membranes via formation of an alpha helical structure in its N-terminal end. We recently demonstrated that either incubation of recombinant A53T mutant alpha-synuclein protein with mitochondria isolated from immortalized midbrainderived dopaminergic neurons or following A53T expression within dopaminergic cells either in culture or in transgenic animals, the protein localizes to the inner mitochondria! membrane (IMM) in the form of oligomers. Mitochondrial localization may be due to A53T's inability to undergo serine 129 (ser-129) phosphorylation as this event, which can be induced by oxidative stress, drives the wildtype protein towards cytoplasmic localization and oligomerization. Localization of A53T to the IMM is accompanied by decreased mitochondrial membrane potential (MMP) and increased mitochondrial autophagy (mitophagy). Decreases in MMP has been shown by others to influence the mitochondrial fission-fusion rate, driving mitochondria towards mitophagy. In aim #1 of this grant application, we propose to: (1) Assess the ability of alphasynuclein ser-129 phosphorylation to determine its subcellular localization and oligomerization (selfinteraction) state with the assistance of Drs. Gibson and Hughes of Components 11 and 6, respectively, (2) Examine the impact of mitochondrial localization on mitochondrial fission-fusion ratios and mitophagy with the assistance of Dr. Nicholls of Component 11, and (3) Examine the effects of alterations in TOR activity on associated mitophagy with the assistance of Dr. Kapahi (Component 2 and co-Pi, Component 9). ' Nuclear translocation of alpha-synuclein selectively into dopaminergic midbrain neurons has been demonstrated following either its overexpression or increased oxidative stress. Nuclear translocation may also be dependent upon the protein's ser-129 phosphorylation state and/or its cleavage as well as its ability to associate with dopaminergic cytoplasmic factors. Nuclear localization appears to result in neurotoxicity via alpha-synuclein's ability to bind histones within the nucleus reducing their acetylation. Reduced histone acetylation could impact on gene transcription. In aim 2 of the application, we propose to assess alphasynuclein for post-translational modifications and interactions with dopaminergic cytosolic factors associated with its nuclear translocation with the assistance of Drs. Gibson and Hughes of Components 11 and 6, respectively. With Dr. Ellerby as part of Component 7, we will also examine the ability of HDAC inhibitors or specific HDAC siRNAs to protect against these effects and with Drs. Vijg and Melov as part of Component 5 the impact of nuclear localization of alpha synuclein on gene transcription.